The present invention relates to novel compounds which bind to the integrin receptor xcex1vxcex23, to pharmaceutical compositions containing such compounds, processes for preparing such compounds, and to methods of using these compounds, alone or in combination with other therapeutic agents.
Integrins are a group of cell surface glycoproteins which mediate cell adhesion and therefore are useful mediators of cell adhesion interactions which occur during various biological processes. Integrins are heterodimers composed of noncovalently linked xcex1 and xcex2 polypeptide subunits. Currently eleven different a subunits have been identified and six different xcex2 subunits have been identified. The various xcex1 subunits can combine with various xcex2 subunits to form distinct integrins.
The integrin identified as xcex1vxcex23 (also known as the vitronectin receptor) has been identified as an integrin which plays a role in various conditions or disease states including tumor metastasis, solid tumor growth (neoplasia), osteoporosis, Paget""s disease, humoral hypercalcemia of malignancy, angiogenesis, including tumor angiogenesis, retinopathy including macular degeneration, arthritis, including rheumatoid arthritis, periodontal disease, psoriasis and smooth muscle cell migration (e.g. restenosis artherosclerosis). Additionally, it has been found that such agents would be useful as antivirals, antifungals and antimicrobials. Thus, compounds which selectively inhibit or antagonize xcex1vxcex23 would be beneficial for treating such conditions.
It has been shown that the xcex1vxcex23 integrin and other xcex1v containing integrins bind to a number of Arg-Gly-Asp (RGD) containing matrix macromolecules. Compounds containing the RGD sequence mimic extracellular matrix ligands so as to bind to cell surface receptors. However, it is also known that RGD peptides in general are non-selective for RGD dependent integrins. For example, most RGD peptides which bind to xcex1vxcex23 also bind to xcex1vxcex25, xcex1vxcex21 and xcex1IIbxcex23. Antagonism of platelet xcex1IIbxcex23 (also known as the fibrinogen receptor) is known to block platelet aggregation in humans. In order to avoid bleeding side-effects when treating the conditions or disease states associated with the integrin xcex1vxcex23, it would be beneficial to develop compounds which are selective antagonists of xcex1vxcex23 as opposed to xcex1IIbxcex23.
The compounds of this invention are therefore selective xcex1vxcex23 integrin antagonist. The present invention includes compounds which inhibit the respective integrins and also includes pharmaceutical compositions comprising such compounds. The present invention further provides for methods for treating or preventing conditions mediated by the xcex1vxcex23 receptor in a mammal in need of such treatment comprising administering a therapeutically effective amount of the compounds of the present invention and pharmaceutical compositions of the present invention. Administration of such compounds and compositions of the present invention inhibits angiogenesis, tumor metastasis, tumor growth, osteoporosis, Paget""s disease, humoral hypercalcemia of malignancy, retinopathy, macular degeneration, arthritis, periodontal disease, smooth muscle cell migration, including restenosis and artherosclerosis, and viral diseases.
Tumor cell invasion occurs by a three step process: 1) tumor cell attachment to extracellular matrix; 2) proteolytic dissolution of the matrix; and 3) movement of the cells through the dissolved barrier. This process can occur repeatedly and can result in metastases at sites distant from the original tumor.
Seftor et al. (Proc. Natl. Acad. Sci. USA, Vol. 89 (1992) 1557-1561) have shown that the xcex1vxcex23 integrin has a biological function in melanoma cell invasion. Montgomery et al., (Proc. Natl. Acad. Sci. USA, Vol. 91 (1994) 8856-60) have demonstrated that the integrin xcex1vxcex23 expressed on human melanoma cells promotes a survival signal, protecting the cells from apoptosis. Mediation of the tumor cell metastatic pathway by interference with the xcex1vxcex23 integrin cell adhesion receptor to impede tumor metastasis would be beneficial.
Brooks et al. (Cell, Vol. 79 (1994) 1157-1164) have demonstrated that antagonists of xcex1vxcex23 provide a therapeutic approach for the treatment of neoplasia (inhibition of solid tumor growth) since systemic administration of xcex1vxcex23 antagonists causes dramatic regression of various histologically distinct human tumors.
The adhesion receptor integrin xcex1vxcex23 was identified as a marker of angiogenic blood vessels in chick and man and therefore such receptor plays a critical role in angiogenesis or neovascularization. Angiogenesis is characterized by the invasion, migration and proliferation of smooth muscle and endothelial cells. Antagonists of xcex1vxcex23 inhibit this process by selectively promoting apoptosis of cells in neovasculature. The growth of new blood vessels, or angiogenesis, also contributes to pathological conditions such as diabetic retinopathy including macular degeneration (Adamis et al., Amer. J. Ophthal., Vol. 118, (1994) 445-450) and rheumatoid arthritis (Peacock et al., J. Exp. Med., Vol. 175, (1992), 1135-1138). Therefore, xcex1vxcex23 antagonists would be useful therapeutic agents for treating such conditions associated with neovascularization (Brooks et al., Science, Vol. 264, (1994), 569-571).
It has been reported that the cell surface receptor xcex1vxcex23 is the major integrin on osteoclasts responsible for attachment to bone. Osteoclasts cause bone resorption and when such bone resorbing activity exceeds bone forming activity it results in osteoporosis (loss of bone), which leads to an increased number of bone fractures, incapacitation and increased mortality. Antagonists of xcex1vxcex23 have been shown to be potent inhibitors of osteoclastic activity both in vitro [Sato et al., J. Cell. Biol., Vol. 111 (1990) 1713-1723] and in vivo [Fisher et al., Endocrinology, Vol. 132 (1993) 1411-1413]. Antagonism of xcex1vxcex23 leads to decreased bone resorption and therefore restores a normal balance of bone forming and resorbing activity. Thus it would be beneficial to provide antagonists of osteoclast xcex1vxcex23 which are effective inhibitors of bone resorption and therefore are useful in the treatment or prevention of osteoporosis.
The role of the xcex1vxcex23 integrin in smooth muscle cell migration also makes it a therapeutic target for prevention or inhibition of neointimal hyperplasia which is a leading cause of restenosis after vascular procedures (Choi et al., J. Vasc. Surg. Vol. 19(1) (1994) 125-34). Prevention or inhibition of neointimal hyperplasia by pharmaceutical agents to prevent or inhibit restenosis would be beneficial.
White (Current Biology, Vol. 3(9)(1993) 596-599) has reported that adenovirus uses xcex1vxcex23 for entering host cells. The integrin appears to be required for endocytosis of the virus particle and may be required for penetration of the viral genome into the host cell cytoplasm. Thus compounds which inhibit xcex1vxcex23 would find usefulness as antiviral agents.
The present invention as a first object provides novel compounds of the following formula (I) 
or a pharmaceutically acceptable salt, prodrug or ester thereof, wherein:
G is selected from the group consisting of 
wherein Q is NH or O and Qxe2x80x2 is H, C1-C6 alkyl, phenyl, or phenyl-C1-C4-alkyl; 
wherein Rxe2x80x2 and Rxe2x80x3 are independently H or C1-C4 alkyl;
B is (CH2)m(CHxe2x95x90CH)pY, wherein m=1,2,3, p=0,1, Y is CH2 or CO.
X is CH2 or Cxe2x95x90O;
R1 is selected from the group consisting of H, C1-C4 alkyl, C1-C4 alkoxy, OH, halogen, CF3;
A is CH2, NH, O, S(O), wherein n is zero, 1 or 2.
R2 is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or C5-C7 monocyclic heteroaryl ring containing one to three heteroatoms selected from O, S, and N, unsubstituted or optionally substituted by one to three substituents independently selected from H, C1-C4 alkyl, C1-C4 alkoxy, OH, halogen, CF3;
R is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C4 alkynyl, aryl or aryl-C1-C4 alkyl.
The present invention includes within its scope all possible isomers, stereoisomers and optical isomers and their mixtures, the chemical and bioprecursors and metabolites of the compounds of formula (I).
It is another object of the invention to provide pharmaceutical compositions comprising compounds of the Formula I. Such compounds and compositions are useful in selectively inhibiting or antagonizing the xcex1vxcex23 integrin and therefore in another embodiment the present invention relates to a method of selectively inhibiting or antagonizing the xcex1vxcex23 integrin. The invention further involves treating or inhibiting pathological conditions associated therewith such as osteoporosis, humoral hypercalcemia of malignancy, Paget""s disease, tumor metastasis, solid tumor growth (neoplasia), angiogenesis, including tumor angiogenesis, retinopathy including macular degeneration and diabetic retinopathy, arthritis, including rheumatoid arthritis, periodontal disease, psoriasis, smooth muscle cell migration and restenosis in a mammal in need of such treatment. Additionally, such pharmaceutical agents are useful as antiviral agents, and antimicrobials.
The present invention relates to a class of compounds represented by the Formula I, described above.
A halogen atom is preferably chlorine or fluorine.
The alkyl, alkoxy, alkenyl and alkynyl groups and the alkylene and alkenylene chains may be branched or straight groups or chains, respectively.
An aryl group is, e.g., an aromatic C6-C20 mono- or poly-nuclear moiety, typically phenyl, naphthyl, unsubstituted or substituted by one to three substituents independently chosen from halogen, hydroxy, CF3, C1-C4 alkyl and C1-C4 alkoxy.
A C5-C7 monocyclic heteroaryl ring is preferably a C5-C6 heteromonocyclic ring, in particular selected from pyridine, pyrazine, pyridazine, pyrimidine, thiophene, pyrrole, pyrazole, imidazole, oxazole and isoxazole.
Accordingly an aralkyl group is e.g. benzyl or phenethyl, in which the phenyl ring is optionally substituted by one to three substituents independently selected from halogen, hydroxy, CF3, C1-C4 alkyl and C1-C4 alkoxy.
A C2-C6 alkenyl group is preferably an allyl group.
A C1-C6 alkyl group is preferably a C1-C4 alkyl group.
A C1-C4 alkyl group is preferably a methyl or ethyl group.
A C2-C4 alkynyl group is preferably an ethynyl group.
A C1-C4 alkoxy group is preferably methoxy, ethoxy, propoxy or butoxy.
Examples of pharmaceutically acceptable salts of the compounds of the invention are either those with inorganic bases, such as sodium, potassium, calcium and aluminum hydroxides, or with organic bases, such as lysine, arginine, N-methyl-glucamine, triethylamine, triethanolamine, dibenzylamine, methylbenzylamine, di-(2-ethyl-hexyl)-amine, piperidine, N-ethylpiperidine, N,N-diethylaminoethylamine, N-ethylmorpholine, xcex2-phenethylamine, N-benzyl-xcex2-phenethylamine, N-benzyl-N,N-dimethylamine and the other acceptable organic amines, as well as the salts with inorganic acids, e.g. hydrochloric, hydrobromic and sulphuric acids and with organic acids, e.g. citric, tartaric, maleic, malic, fumaric, trifluoroacetic, methanesulphonic and ethanesulphonic acids.
As stated above, the present invention also includes within its scope pharmaceutically acceptable bio-precursors (otherwise known as pro-drugs) of the compounds of formula (I), i.e. compounds which have a different formula to formula (I) above, but which nevertheless upon administration to a human being are converted directly or indirectly in vivo into a compound of formula (I).
Preferred compounds of the invention are those, in formula (I), wherein R, R1, X and A are as defined above;
B is (CH2)m(CHxe2x95x90CH)pY, wherein m=1,2,3, p=0 and Y is CH2;
R2 is a phenyl, naphthyl, pyridine, pyrazine, pyridazine, pyrimidine, thiophene, pyrrole, pyrazole, imidazole, oxazole and isoxazole unsubstituted or optionally substituted as defined above; and G is selected from the group consisting of 
wherein Q is NH or O and Qxe2x80x2 is H, C1-C6 alkyl, phenyl, or phenyl- C1-C4-alkyl; 
and the pharmaceutically acceptable salts, prodrugs and esters thereof.
Most preferred compounds of the invention are those wherein, in formula (I), B, X, R1, A and R are as defined above
R2 is a phenyl, thiophene, oxazole, isoxazole, or a pyridine ring unsubstituted or optionally substituted as defined above; and G is selected from the group consisting of 
and the pharmaceutically acceptable salts, prodrugs and esters thereof.
Examples of specific preferred compounds according to the invention are the following:
3-phenyl-N-{4-[2-(2-pyridinylamino)ethyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-beta-alanine;
3-phenyl-N-{4-[3-(2-pyridinylamino)propyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-beta-alanine;
3-phenyl-N-{4-[4-(2-pyridinylamino)butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-beta-alanine;
N-{4-[2-(1H-imidazol-2-ylamino)ethyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-phenyl-beta-alanine;
N-{4-[3-(1H-imidazol-2-ylamino)propyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-phenyl-beta-alanine;
N-{4-[2-(1H-imidazol-2-ylamino)butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-phenyl-beta-alanine;
3-(3-pyridinyl)-N-{4-[2-(2-pyridinylamino)ethyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-beta-alanine;
3-(3-pyridinyl)-N-{4-[3-(2-pyridinylamino)propyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-beta-alanine;
3-(3-pyridinyl)-N-{4-[4-(2-pyridinylamino)butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-beta-alanine;
N-{4-[2-(1H-imidazol-2-ylamino)ethyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-(3-pyridinyl)-beta-alanine;
N-{4-[3-(1H-imidazol-2-ylamino)propyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-(3-pyridinyl)-beta-alanine;
N-{4-[4-(1H-imidazol-2-ylamino)butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-(3-pyridinyl)-beta-alanine;
N-{3-oxo-4-[2-(2-pyridinylamino)ethyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-phenyl-beta-alanine;
N-{3-oxo-4-[3-(2-pyridinylamino)propyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-phenyl-beta-alanine;
N-{3-oxo-4-[4-(2-pyridinylamino)butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-phenyl-beta-alanine;
N-{4-[2-(1H-imidazol-2-ylamino)ethyl]-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-phenyl-beta-alanine;
N-{4-[3-(1H-imidazol-2-ylamino)propyl]-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-phenyl-beta-alanine;
N-{4-[4-(1H-imidazol-2-ylamino)butyl]-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-phenyl-beta-alanine;
N-{3-oxo-4-[2-(2-pyridinylamino)ethyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-(3-pyridinyl)-beta-alanine;
N-{3-oxo-4-[3-(2-pyridinylamino)propyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-(3-pyridinyl)-beta-alanine;
N-{3-oxo-4-[4-(2-pyridinylamino)butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-(3-pyridinyl)-beta-alanine;
N-{4-[2-(1H-imidazol-2-ylamino)ethyl]-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-(3-pyridinyl)-beta-alanine;
N-{4-[3-(1H-imidazol-2-ylamino)propyl]-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-(3-pyridinyl)-beta-alanine;
N-{4-[4-(1H-imidazol-2-ylamino)butyl]-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-(3-pyridinyl)-beta-alanine;
3-({3-oxo-4-[3-(1H-imidazol-2-ylamino)propyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}oxy)-3-phenylpropanoic acid;
3-({3-oxo-4-[4-(2-pyridinylamino)butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}oxy)-3-phenylpropanoic acid;
3-({3-oxo-4-[4-(1H-imidazol-2-ylamino)butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}oxy)-3-phenylpropanoic acid;
3-({3-oxo-4-[3-(2-pyridinylamino)propyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}oxy)-3-(3-pyridinyl)propanoic acid;
3-({3-oxo-4-[4-(2-pyridinylamino)butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}oxy)-3-(3-pyridinyl)propanoic acid;
3-({3-oxo-4-[3-(1H-imidazol-2-ylamino)propyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}oxy)-3-(3-pyridinyl)propanoic acid;
3-({3-oxo-4-[4-(1H-imidazol-2-ylamino)butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}oxy)-3-(pyridinyl)propanoic acid;
3-({3-oxo-4-[4-(2-pyridinylamino)butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}sulfanyl)-3-phenylpropanoic acid;
3-({3-oxo-4-[3-(1H-imidazol-2-ylamino)propyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}sulfanyl)-3-phenylpropanoic acid;
3-({3-oxo-4-[4-(1H-imidazol-2-ylamino)butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}sulfanyl)-3-phenylpropanoic acid;
3-({3-oxo-4-[3-(2-pyridinylamino)propyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}sulfanyl)-3-(3-ppyridinyl)propanoic acid;
3-({3-oxo-4-[4-(2-pyridinylamino)butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}sulfanyl)-3-(3-pyridinyl)propanoic acid;
3-({3-oxo-4-[3-(1H-imidazol-2-ylamino)propyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}sulfanyl)-3-(3-pyridinyl)propanoic acid;
3-({3-oxo-4-[4-(1H-imidazol-2-ylamino)butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}sulfanyl)-3-(3-pyridinyl)propanoic acid;
4-{3-oxo-4-[4-(pyridin-2-ylamino)-butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-phenylbutanoic acid;
4-{3-oxo-4-[3-(1H-imidazol-2-ylamino)-propyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-phenylbutanoic acid;
4-{3-oxo-4-[4-(1H-imidazol-2-ylamino)-butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-phenylbutanoic acid;
4-{3-oxo-4-[4-(pyridin-2-ylamino)-butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-(3-pyridinyl)butanoic acid;
4-{3-oxo-4-[3-(1H-imidazol-2-ylamino)-propyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-(3-pyridinyl)butanoic acid;
4-{3-oxo-4-[4-(1H-imidazol-2-ylamino)-butyl]-3,4-dihydro-2H-1,4-benzoxazin-7-yl}-3-(3-pyridinyl)butanoic acid;
A further object of the present invention is to provide a compound of formula (I) as defined above or a pharmaceutically acceptable salt, prodrug or ester thereof, for use in a method of treatment of the human or animal body by therapy, in particular for treating conditions mediated by the xcex1vxcex23 integrin.
The object of the present invention is also to provide a pharmaceutical composition comprising a pharmaceutically acceptable carrier and/or diluent and as an active principle a compound of formula (I), as herein defined, or a pharmaceutically acceptable salt thereof.
The present invention also provides the use of a compound of formula (I), as defined above, in the preparation of a medicament having xcex1vxcex23 integrin inhibiting or antagonizing activity.
The present invention also provides a method for treating conditions mediated by the xcex1vxcex23 integrin in a mammal, including humans, in need of such treatment comprising administering to said mammal an effective xcex1vxcex23 inhibiting or antagonizing amount of a compound of formula (I) as defined above or a pharmaceutically acceptable salt thereof
More specifically, the present invention provides a method for inhibiting bone resorption, treating osteoporosis, inhibiting humoral hypercalcemia of malignancy, treating Paget""s disease, inhibiting tumor metastasis, inhibiting neoplasia (solid tumor growth), inhibiting angiogenesis including tumor angiogenesis, treating diabetic retinopathy, inhibiting arthritis, psoriasis and periodontal disease, and inhibiting smooth muscle cell migration including restenosis.
The compounds of the invention and the salts thereof can be prepared by or in analogy to the processes described hereafter. Accordingly, compounds of formula I and the salts thereof, can be for instance obtained by a process as described here below.
A compound of formula I wherein G is as defined above and Q is different from oxygen; X, B, A, R, R1 and R2 are as defined above can be obtained by reacting a compound of formula II 
wherein B, X, A, R, R1 and R2 are as defined above, G1 is represented by G, as defined above under a) to g) wherein Q is different from oxygen and one or more nitrogens are protected with suitable protective groups, such as, for instance, carbobenzyloxy or t-butoxycarbonyl, removable with a suitable reducing agent, or with an acid, such as trifluoroacetic acid or hydrochloric or hydrobromic acid.
A compound of formula II can be obtained by:
1) reacting a compound of formula III 
wherein G1, X, B, R, R1 and R2 are as defined above and A is NH, with a suitable reducing agent, such as an alkali metal borohydride or an alkali metal cyanoborohydride thus obtaining a compound of formula II wherein G1, X, B, R, R1 and R2 are as defined above and A is NH. A compound of formula III, wherein G1, B, X, R, R1 and R2 are as defined above and A is NH, can, in turn, be obtained by reacting a compound of formula IV 
wherein G1, B, X, and R1 are as defined above and A is NH2, with a compound of formula V 
wherein R and R2 are as defined above; or
2) Reacting a compound of formula IV wherein G1, B, X, and R1 are as defined above and A is SH, with a compound of formula VI 
wherein R and R2 are as defined above, thus obtaining a compound of formula II wherein G, X, B, R, R1 and R2 are as defined above, and A is S; or
3) Reacting compound of formula IV where G1, B, X and R1 are as defined above and A is OH with a compound of formula VII 
wherein R and R2 are as defined above, and W is Cl, Br, I, OH or OSO2C1-C4-alkyl or OSO2Aryl, thus obtaining a compound of formula II wherein G, X, B, R, R1 and R2 are as defined above, and A is O; or
4) Reacting a compound of formula II, wherein G1, X, B, R, R1 and R2 are as defined above, and A is S, with an oxydizing agent such as NalO4, oxone, H2O2 or a peracid, thus obtaining a compound of formula II wherein G1, X, B, R1, R2 and R are as defined above and A is S(O)n wherein n is 1 or 2, i.e. A is a sulfoxide or sulfone group; or
5) Reacting a compound of formula VIII 
wherein X, R, R1, R2 are as defined above, and A is CH2, with a compound of formula IX
G1xe2x80x94Bxe2x80x94Wxe2x80x83xe2x80x83IX 
wherein G1, B and W are as defined above, thus obtaining a compound of formula II wherein G1, X, B, R1, R2 and R are as defined above and A is CH2. A compound of formula VIII, as defined above, can, in turn, be obtained by reacting a compound of formula X 
wherein X and R1 are as defined above, A is CH2, W is selected from Cl or Br and Z1 is a suitable amide protecting group, such as a p-methoxy-benxyl group, with magnesium followed by addition of the resulting compound to a compound of formula VI, wherein R and R2 are as described above, in the presence of suitable metal salts, like copper salts, followed by removal of Z1.
A compound of formula IV, as defined above wherein A is NH2, can be obtained by reduction of a compound of formula XI 
wherein G1, X, B and R1 are as defined above.
A compound of formula IV, wherein G1, X, B and R1 are as defined above and A is SH or OH can be obtained by a compound of formula XII 
wherein, X, B and R1 are as defined above, A is S or O and Z2 is a suitable protecting group for a xe2x80x94OH or a xe2x80x94SH moiety, such as methyl, p-methoxybenzyl, t-butyldimethylsilyl, p-nitrobenzyl, by removal of the Z2 protecting group.
A compound of formula XI can be obtained by reacting a compound of formula XIII 
wherein X and R1 are as defined above, with a compound of formula IX, as defined above.
A compound of formula XII can be obtained by reacting a compound of formula XIV 
wherein A, X, R1, and Z2 are as defined above, with a compound of formula IX, as defined above.
A compound of formula I, wherein B, X, R, R1, R2 are as defined above, A is CH2, O or S, and G is as defined under a) wherein Q is oxygen, can be obtained by reacting a compound of formula XV 
wherein B, X, R, R1, R2 are as defined above and A is CH2, O or S, with an inorganic cyanate, such as sodium or potassium or ammonium cyanate, or with an isocyanate of formula Qxe2x80x2NCO, wherein Qxe2x80x2 is as described above.
A compound of formula I, wherein B, X, R, R1, R2 are as defined above, A is CH2, O or S, and G is as defined under a) wherein Q is different from oxygen, or under b) and c), and Qxe2x80x2 is hydrogen, can be obtained by reacting a compound of formula XV with a suitable guanylating agent, such as, for instance, N,Nxe2x80x2-di-t-butoxycarbonyl-Nxe2x80x3-triflylguanidine or 2-methylthio-2-imidazoline hydriodide or 2-methylthio-1,4,5,6-tetrahydropyrimidine hydriodide, followed by removal of the protecting groups in the case where the guanilating agent is N,Nxe2x80x2-di-t-butoxycarbonyl-Nxe2x80x3-triflylguanidine.
A compound of formula XV, as defined above, can be obtained by reaction of a compound of formula VIII, wherein X, R, R1, R2 are as defined above, and A is CH2, O or S, with a compound of formula XVI
Z3xe2x80x94NHxe2x80x94Bxe2x80x94Wxe2x80x83xe2x80x83XVI 
wherein B and W are as defined above and Z3 is a suitable amino protecting group such as, for instance, carbobenzyloxy or t-butoxycarbonyl, followed by removal of Z3.
A compound of formula I, wherein B, X, R, R1, R2 are as defined above, A is NH, and G is as defined under a) wherein Q is oxygen, can be obtained by reacting a compound of formula XVII 
wherein B and R1 are as defined above, G is as defined under a) wherein Q is oxygen, and A is NH2, with a compound of formula V, followed by reduction of the product with an alkali metal borohydride or an alkali metal cyanoboro-hydride. A compound of formula XVII, as defined above, can be obtained in analogy to the procedures described for the synthesis of compounds of formula IV, provided that G, as defined above, is substituted for G1.
A compound of formula I, wherein B, X, R, R1, R2 are as defined above, A is NH, and G is as defined under a) wherein Q is different from oxygen, or under b) or c), and Qxe2x80x2 is hydrogen, can be obtained by reacting a compound of formula XV, wherein B, X, R, R1 and R2 are as defined above, and A is NH, with a suitable guanilating agent, such as, for instance, N,Nxe2x80x2-di-t-butoxy-carbonyl-Nxe2x80x3-triflylguanidine or 2-methylthio-2-imidazoline hydriodide or 2-methylthio-1,4,5,6-tetrahydropyrimidine hydriodide, followed by removal of the protecting groups in the case where the guanilating agent is N,Nxe2x80x2-di-t-butoxy-carbonyl-Nxe2x80x3-triflylguanidine.
A compound of formula XV, wherein B, X, R, R1 and R2 are as defined above, and A is NH, in turn, can be obtained by reacting a compound of formula XIII, as defined above, with a compound of formula XVI, as defined above, followed by: a) reduction of the nitro group, b) reaction with a compound of formula V, as defined above, c) reduction of the resulting compound with an alkali metal borohydride or an alkali metal cianoborohydride, d) removal of the protectin group Z3.
A compound of formula X or XIII or XIV, as defined above, can be prepared according to general methods described in the literature.
Additionally, a compound of formula I can be obtained by a process which comprises:
a) converting a compound of formula (I) into another compound of formula (I), or separating a single isomer of a compound of formula (I) from a mixture thereof, or converting a compound of formula (I) into a salt thereof, or converting a salt of a compound of formula (I) into a free compound of formula (I);
b) reacting a compound of formula I wherein G, X, B, A R1 and R2 are as defined above and R is different from hydrogen with aqueous acids or aqueous bases thus obtaining a compound of formula I wherein R is hydrogen;
The compounds described above can be prepared as exemplified in the following procedures.
In a typical procedure, a compound of formula I, as defined above, can be obtained by treatment of a compound of formula II, as defined above, with hydrogen in the presence of a suitable catalyst, such as palladium on carbon, in a solvent like a lower alcohol or dioxane, or by treatment with trifluoroacetic acid or hydrobromic acid in acetic acid, followed by isolation of the product by chromatography.
In a typical procedure for the preparation of a compound of formula III, as defined above, a compound of formula IV, as defined above, a keto-ester of formula V and anhydrous calcium sulfate and catalytic acetic acid are mixed in an anhydrous alcohol, such as ethanol, and refluxed for 24 to 96 hours. The compound is isolated by column chromatography. The reduction of a compound of formula III with sodium cyanoborohydride in ethanol provides a compound of formula II, as defined above and wherein A is NH.
In a typical procedure for the preparation of a compound of formula II, as defined above and wherein A is S, a compound of formula IV, as defined above and wherein A is SH, is added to a solution of a compound of formula VI, as defined above, in a solvent like tetrahydrofuran or dichloromethane, in the presence of a small amount of an organic base such as diazabicycloundecene (DBU). The reaction is allowed to proceed from 12 to 48 hours, at a temperature from about 20 to about 40xc2x0 C., and the product isolated by column chromatography.
In a typical procedure for the preparation of a compound of formula II, as defined above and wherein A is O, to a dichloromethane or tetrahydrofuran solution containing a compound of formula IV, as defined above and wherein A is OH, triphenylphosphine and a compound of formula VII, as defined above and wherein W is OH, a solution of an alkyl azodicarboxylate in dichloromethane or tetrahydrofuran is added and the mixture stirred at a temperature from room temperature to about 40xc2x0 C. for 1 to 6 hours. The product is then isolated by column chromatography. The procedure described above is similar to procedures disclosed in the literature (see, for instance, KRCHNAK, V., FLEGELOVA, Z., WEICHSEL, A. S., LEBL, M. Tetrahedron Letters 1995, 36, 6193-6196; or BROWN, R. F. C., JACKSON, W. R., McCARTHY, T. D. Tetrahedron 1994, 18, 5469-5488).
In a typical procedure for the preparation of a compound of formula II, as defined above and wherein A is SO2, a compound of formula II, as defined above and wherein A is S, is treated with 30% aqueous H2O2 in an alcohol as a solvent, or with NalO4 in water and methanol, or acetone and water, (as described, for instance, in BEIER, C., SCHAUMANN, E. Synthesis 1997, 11, 1296-1300).
In a typical procedure for the preparation of a compound of formula II, as defined above and wherein A is SO, a compound of formula II, as defined above and wherein A is S, is treated with oxone in a mixture methanol/water as a solvent (as described, for instance, in HINTERBERGER, S., HOFER, O., GREGER, H. Tetrahedron 1998, 54, 487-496).
In a typical procedure for the preparation of a compound of formula II, as defined above and wherein A is CH2, a Grignard reagent obtained from a compound of formula X, as defined above and wherein W is Cl or Br and P1 is p-methoxybenzyl, is added to a mixture of cuprous iodide (Cul) and dry tetramethylene diamine (TMEDA) in dry tetrahydrofuran at a temperature of about xe2x88x9278xc2x0 C. The mixture is then stirred at about xe2x88x9278xc2x0 C. for between 15 minutes to 1 hours, then a tetrahydrofuran solution of chlorotrimethyl silane and a compound of formula IV, as defined above, is added and the temperature allowed to rise to about xe2x88x9230xc2x0 C. After a period of 12 to 36 hours, the reaction is treated with a saturated aqueous solution of ammonium chloride and ammonium hydroxide and extracted with a suitable solvent, such as dichloromethane, chloroform, diethylether, ethyl acetate and the product isolated by column chromatography (see, for instance, VAN HEERDEN, P. S., BEZUIDENHOUDT, B. C. B., FERREIRA, D. Tetrahedron 1996, 52, 12313-12322; or VAN HEERDEN, P. S., BEZUIDENHOUDT, B. C. B., FERREIRA, D. J. Chem. Soc. Perk. Trans. 1, 1997, 1141-1146). Then, the p-methoxybenzyl group is removed by catalytic hydrogenolysis, using a supported metal catalyst, such as palladium on carbon in a solvent like methanol or ethanol, to give a compound of formula VIII, as defined above and wherein A is CH2. A compound of formula VIII, as defined above, is then reacted with a compound of formula IX, as defined above and wherein W is typically OSO2CH3, in the presence of an organic base, such as potassium hexamethyldisilazide in a solvent like tetrahydrofuran or dimethylformamide or mixtures thereof, at a temperature from room temperature to about 100xc2x0 C. for 1 to 6 hours, the product isolated by column chromatography thus giving a compound of formula II, as defined above and wherein A is CH2.
In typical procedure for the preparation of a compound of formula IV, as defined above and wherein A is NH2, a compound of formula XI, as defined above, is reduced by treatment with a mixture of titanium tetrachloride and tin dichloride in a solvent like tetrahydrofuran or diisopropyl ether or diethyl ether, at a temperature from about 20 to about 40xc2x0 C. and under an atmosphere of an inert gas. A compound of formula XI, as defined above, can be obtained by treatment of a compound of formula XIII, as defined above, with a compound of formula IX, as defined above and wherein W is typically OSO2CH3, in the presence of an organic base, such as potassium hexamethyldisilazide in a solvent like tetrahydrofuran or dimethylformamide or mixtures thereof, at a temperature from room temperature to about 100xc2x0 C. for 1 to 6 hours, and the product isolated by column chromatography.
In typical procedure for the preparation of a compound of formula XII, as defined above and wherein A is O or S and Z2 is a protecting group such as methyl, ethoxymethyl, p-methoxybenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, p-nitrobenzyl or other suitable protecting group (see: GREENE, T. W., WUTS, P. G. M. Protective group in organic synthesis, Wiley, 1999), a compound of formula XIV, as defined above, is reacted with a compound of formula IX, as defined above and wherein W is typically OSO2CH3, in the presence of an organic base, such as potassium hexamethyldisilazide in a solvent like tetrahydrofuran or dimethylformamide or mixtures thereof, at a temperature from room temperature to about 100xc2x0 C. for 1 to 6 hours.
In typical procedure for the preparation of a compound of formula I, wherein B, X, R, R1, R2 are as defined above, A is CH2, O or S, and G is as defined under a) wherein Q is oxygen and Qxe2x80x2 is hydrogen, a compound of formula XV, as defined above, is reacted with a cyanate salt, as e.g. an ammonium or sodium or potassium salt, in solvent such as acetic acid or water, at a temperature from about 50 to about 100xc2x0 C., for 2 to 12 hours (see, as an example, Organic Synthesis vol. IV, p. 49, 1963).
In typical procedure for the preparation of a compound of formula I, wherein B, X, R, R1, R2 are as defined above, A is CH2, O or S, and G is as defined under a) wherein Q is oxygen and Qxe2x80x2 is different from hydrogen, a compound of formula XV, as defined above, is reacted with an isocyanate of formula Qxe2x80x2NCO, wherein Qxe2x80x2 is as defined above, in a solvent like dichloromethane, acetonitrile, tetrahydrofuran, dioxane or toluene, in the presence of triethylamine, at a temperature from room temperature to about 100xc2x0 C. and for 4 to 24 hours. The resulting compound is isolated by column chromatography.
In a typical procedure for the preparation of a compound of formula I, wherein B, X, R, R1, R2 are as defined above, A is CH2, O or S, and G is as defined under a) wherein Q is different from oxygen, or under b) and c), and Qxe2x80x2 is hydrogen, a compound of formula XV is treated with a suitable guanylating agent, like N,Nxe2x80x2-di-t-butoxycarbonyl-Nxe2x80x3-triflylguanidine or 2-methylthio-2-imidazoline hydriodide or 2-methylthio-1,4,5,6-tetrahydropyrimidine hydriodid in a solvent like dichloromethane, tetrahydrofuran, dioxane or a lower alcohol, at a temperature from room temperature to reflux, for 24 to 72 hours, followed by evaporation of the solvent and isolation of the product by chromatography. In typical procedure for the preparation of a compound of formula XV, as defined above, a compound of formula VIII, wherein X, R1, R2 are as defined above, R is different from hydrogen and A is CH2, O or S, with a compound of formula XVI, as defined above and wherein W is Br or OSO2CH3, in the presence of an organic base, such as potassium hexamethyldisilazide in a solvent like tetrahydrofuran or dimethylformamide or mixtures thereof, at a temperature from room temperature to 100xc2x0 C. for 1 to 6 hours. The resulting compound is isolated by column chromatography, and the protecting group Z3 is then removed following the standard procedures described in the literature (see: GREENE, T. W., WUTS, P. G. M. Protective group in organic synthesis, Wiley, 1999).
In typical procedure for the preparation of a compound of formula I, wherein B, X, R, R1, R2 are as defined above, A is NH, G is as defined under a) wherein Q is oxygen and Qxe2x80x2 is as defined above, a compound of formula XVII, as defined above, is mixed with a keto-ester of formula V in the presence of anhydrous calcium sulfate and catalytic acetic acid, in a solvent like an anhydrous alcohol, such as ethanol, and refluxed for 24 to 96 hours. The compound is isolated by column chromatography, then is treated with sodium cyanoborohydride in ethanol thus giving a compound of formula I, as defined above. A compound of formula XVII, as defined above, can be obtained using the same procedure described above for the synthesis of compounds of formula IV, provided that G, as defined under a), is substituted for G1, and wherein A is NH2.
In typical procedure for the preparation of a compound of formula I, wherein B, X, R, R1, R2 are as defined above, A is NH, G is as defined under a) wherein Q is different from oxygen, or under b) or c), and Qxe2x80x2 is hydrogen, a compound of formula XV, wherein B, X, R, R1 and R2 are as defined above, and A is NH is reacted with a suitable guanylating agent, like N,Nxe2x80x2-di-t-butoxycarbonyl-Nxe2x80x3-triflylguanidine or 2-methylthio-2-imidazoline hydriodide or 2-methylthio-1,4,5,6-tetrahydropyrimidine hydriodid in a solvent like dichloromethane, tetrahydrofuran, dioxane or a lower alcohol, at a temperature from room temperature to reflux, for 24 to 72 hours, followed by evaporation of the solvent and isolation of the product by chromatography. In the case where the guanilating agent is N,Nxe2x80x2-di-t-butoxycarbonyl-Nxe2x80x3-triflylguanidine, the product is then treated with trifluoroacetic acid, to remove the nitrogen protecting groups, the trifluoroacetic acid is evaporated to afford the desired compound. A compound of formula XV, as defined above, can be obtained using the synthetic sequence described before and applying some of the experimental procedures described before.
In a typical procedure, a compound of formula I, as defined above and wherein R is hydrogen, is obtained by treatment of a compound of formula I, as defined above and wherein R is different form hydrogen, with a mixture of an aqueous acid, like hydrochloric acid, and a lower alcohol or dioxane, at a temperature from room temperature to about 40xc2x0 C., for 1 to 24 hours.
Alternatively, a compound of formula I, as defined above and wherein R is different form hydrogen, is treated with an aqueous base, like sodium or lithium or potassium hydroxide, in a solvent like methanol or ethanol or dioxane, at a temperature from room temperature to about 40xc2x0 C., for 1 to 24 hours. The solution is then treated with an acid and the compound filtered.
A compound of formula X or XIII or XIV, as defined above, can be obtained according to general synthetic methods for 1,4-benzoxazines as described, for instance, in METHODS OF ORGANIC CHEMISTRY (HOUBEN-WEIL), volume E 9a, pp. 141-177, George Thieme Verlag, Stuttgart 1997, or by suitable modifications of such methods as known to those skilled in the art. Also the optional salification of a compound of formula (I) as well as the conversion of a salt into the free compound and the separation of a mixture of isomers into the single isomers may be carried our by conventional methods. For example, the separation of optical isomers may be carried out by salification with an optically active base or acid and by subsequent fractional crystallisation of the diastereoisomeric salts, followed by recovering of the optically active isomeric acids or bases, respectively.
When in the compound of formula (I), and in the intermediate products thereof, groups are present which need to be protected before submitting them to the here-above illustrated reactions, they may be protected before the reactions take place and then deprotected, according to methods well known to those skilled in the art.
The compounds of formula (I) and the pharmaceutically acceptable salts thereof are herein defined as the xe2x80x9ccompounds of the present inventionxe2x80x9d, the xe2x80x9ccompounds of the inventionxe2x80x9d and/or the xe2x80x9cactive principles of the pharmaceutical compositions of the inventionxe2x80x9d.
The following examples describe the invention without limiting it.